Imaging SPR apparatus

ABSTRACT

A two-dimensional imaging surface plasmon resonance (SPR) apparatus for optical surface analysis of a sample area on a sensor surface is disclosed. The apparatus comprises a sensor surface layer of a conductive material that can support a surface plasmon, such as a free electron metal, e.g. gold, silver or aluminum, a source of electromagnetic beams of two or more wavelengths that illuminate a two-dimensional surface area from either the front or the backside of the sensor surface layer, and a detector for simultaneous, or pseudo simultaneous, detection of two or more wavelengths of reflected intensities from the two-dimensional surface area, providing two or more two-dimensional images of the surface area, the two-dimensional images being a function of the effective refractive index at each point on the surface area. The two-dimensional images put together result in a color image. The apparatus is suitable for use in biological, biochemical, chemical and physical testing.

The present application is the U.S. national phase of internationalapplication number PCT/SE01/00530, filed Mar. 14, 2001, which claims thebenefit of U.S. Provisional Application No. 60/189,084, filed Mar. 14,2000.

The present invention relates to an apparatus for optical surfaceanalysis of a sample area on a sensor surface. The invention isparticularly concerned with a two-dimensional imaging surface plasmonresonance (SPR) apparatus suitable for use in biological, biochemical,chemical and physical testing.

BACKGROUND OF THE INVENTION

There is an interest in surface sensitive techniques for quantifyingmolecular interactions. Properties that are of interest are e.g.concentration of free analyte in solution, surface concentration ofmolecules on sensor surface, reaction kinetics between interactingsubstances, affinity of said substances, allosteric effects or epitopemappings. Examples of interacting substances are antigen-antibody,protein-protein, receptor-ligand, DNA-DNA, DNA-RNA, peptides-proteins,carbohydrates-proteins, glycoproteins-proteins, etc.

There are many techniques that are suitable for this task, e.g. surfaceplasmon resonance (SPR), resonant mirror, grating couplers,interferometers, surface acoustic wave (SAW), Quartz CrystalMicrobalance (QCM) etc. So far, SPR is the dominating technique.

Areas of application are e.g. measurement of concentration of substancesin biological research, biochemistry research, chemical research,clinical diagnosis, food diagnostics, environmental measurements, etc.Kinetic measurements can be used to determine rate constants as k_(on)and k_(off). Affinity measurements can be used to determine equilibriumassociation (K_(A)) or dissociation (K_(D)) constant as well as avidity.

SPR is a well-known phenomenon that consists of a bond electromagneticwave, due to oscillations of electrons at the interface of a plasma. Thesurface plasmon can only exist at an interface between said plasma (e.g.a metal) and a dielectricum. A change in the optical constants of thedielectricum will change the propagation constant of the surfaceplasmon. The surface plasmon can be excited by light if the propagationconstant of the light parallel to the interface is equal to, or closeto, the propagation constant of the surface plasmon. Normally one usesthe Kretschmann configuration [1] where a thin metallic film is appliedon a prism, having a higher refractive index than the measured sample.The surface plasmon is then evanescently excited under total internalreflection, i.e. at an incident angle, normal to the surface, largerthan the critical angle. At a certain incident angle, the component ofthe wave vector parallel to the surface meets the real part of thecomplex wave vector for a surface plasmon, and hence the light willcouple into the surface plasmon and propagate at the interface betweensaid plasma and said dielectricum. The surface plasmon will reradiateinto the prism, and for a certain thickness of said plasma a destructiveinterference occur, leading to zero or close to zero intensity ofreflected light. For a smooth surface of said plasma, coupled light willbe absorbed in said plasma and generate heat.

When molecules bind close to the interface (within the probe depth ofthe surface plasmon) the interaction can be detected by a shift in theresonance condition of the surface plasmon. This can be detected as ashift in a reflected light intensity.

The SPR sensor can be used in an imaging mode, also denoted microscopy.This was at first proposed by Yeatman in 1987 [2]. Other setups areproposed by Bengt Ivarsson EP958494A1: ANALYTICAL METHOD AND APPARATUS[3, 4], or GWC Instruments SPRimager [5]. The latter utilizes manywavelengths in a non-simultaneous manner.

The surface plasmon resonance (SPR) phenomenon was already described in1959 [6] and SPR apparatuses for thin adlayer analysis have beenthoroughly described since 1968 [1, 7]. SPR setups for biosensing wereused for the first time in 1983 [8] and for imaging applications in 1987[2, 9]. With imaging SPR, also denoted SPR microscopy, new applicationsarise, e.g., label free—real time—multi spot biochemical analyses [10,11], which can increase the throughput tremendously. The pioneering workon imaging SPR was undertaken by Knoll et al., who investigated surfacespatterned with Langmuir-Blodgett films [12, 13]. They also investigatedthe physical aspects of the technique, including lateral resolution[14], and proposed different setups, e.g. the rotating grating coupler[15].

There are in principal three different ways to measure changes in theSPR propagation constant. First, by measuring the reflected intensity(reflectance) at a flank of the SPR dip at a certain wavelength andincident angle. Second, by measuring the intensity of the reflectedlight versus the angle of incident light (angular interrogation). Third,by measuring the intensity of reflected light for different wavelengthsat a certain incident angle (wavelength interrogation).

For zero-dimensional SPR (measurement of a single spot) said angular orwavelength interrogation requires at least a one-dimensional (linear)detector to make an instant measurement of the position of an SPR dip.For one-dimensional SPR (measurement of a single line) said angular orwavelength interrogation requires at least a two-dimensional (matrix)detector to make an instant measurement of the position of an SPR dip.In this case one dimension is used for the length scale (real image) andone dimension is used for the dip (either angle or wavelength). Iftwo-dimensional SPR-measurement is performed, normally a dip cannot beresolved, i.e. one can normally only make an intensity measurement witha two-dimensional detector, i.e. for the two length scales. This meansthat only a limited portion of the dynamic range (effective refractiveindex of the sample) can be measured, due to the limited extension ofthe SPR-dips (in either angel or wavelength). Only at a small range willthe slope of the SPR-dip be high, which means that there will be alimited range of high sensitivity.

To overcome these drawbacks the present invention provides atwo-dimensional imaging surface plasmon resonance apparatus wherein aset of wavelengths can simultaneously (or pseudo-simultaneously) beused, e.g. by using a multi-wavelength light source and a color camera.

SHORT DESCRIPTION OF THE INVENTION

A new multi-wavelength surface plasmon resonance (SPR) apparatus forimaging applications is presented. It can be used for biosensing, e.g.,for monitoring of chemical and biological reactions in real time withlabel free molecules. A set-up with a fixed incident angle in theKretschmann configuration with gold as the supporting metal isdescribed, both theoretically and experimentally. Simulations of thesensor response based on independently recorded optical (ellipsometric)data of gold show that the sensitivity for 3-dimensional recognitionlayers (bulk) increases with increasing wavelength. For 2-dimensionalrecognition layers (adlayer) maximum sensitivity is obtained within alimited wavelength range. In this situation, the rejection of bulkdisturbances, e.g. emanating from temperature variations, decreases withincreasing wavelength. For SPR imaging, the spatial resolution decreaseswith increasing wavelength. Hence, there is always a compromise betweenspatial resolution, bulk disturbance rejection and sensitivity. Mostimportantly, by simultaneously using multiple wavelengths, it ispossible to maintain a high sensitivity and accuracy over a largedynamic range. Furthermore, our simulations show that the sensitivity isindependent of the refractive index of the prism.

The main advantages of the invention are:

Improvement of the performance of imaging surface plasmon resonance(SPR).

By simultaneously using two or more wavelengths, both sides of theSPR-dip can be tracked, and hence dip width and dip depth changes can bedetected. This will enhance both the accuracy and precision of themeasurement. Absorbing substances (e.g. colloid gold) will induce dipwidth and dip depth changes.

By simultaneously using two or more wavelengths the measuring range canbe extended.

By simultaneously using two or more wavelengths a high sensitivity canbe obtained for a larger measuring range.

By using two or more wavelengths, different points on the sensor surfacewith different effective refractive indices can be measuredsimultaneously with a high sensitivity, high accuracy, and a highprecision.

The simultaneous use of two or more wavelengths will not only improvesensitivity, accuracy, and precision, but will also improve the speed ofanalysis, i.e. a higher throughput is obtainable.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a multi-wavelength imaging SPR setup. The parameters areexplained in the text.

FIG. 1 b shows schematically the apparatus with analog to digitalconverter and computer, and a flow cell with corresponding flow system(pump etc).

FIG. 2 a shows the sensing unit in Kretschmann (back side illumination)configuration containing a prism, metal film and surface chemistry.

FIG. 2 b shows a patterned sensor surface, with 6×7=42 measuring spots.

FIG. 2 c shows a flow cell attached to the sensing unit.

FIG. 2 d shows a version with exchangeable sensing chips.

FIG. 3 shows a sensing unit in the Otto configuration (front sideillumination)

FIG. 4 shows an instrument with grating coupling

FIG. 5 shows a multi-detector arrangement.

FIG. 6 shows an imaging system.

FIG. 7 shows that it is possible to change incident angle.

FIG. 8 a shows a setup with interchangeable filters (pseudosimultaneously)

FIG. 8 b shows a rotating filter wheel.

FIG. 9 shows an experimental setup with a sophisticated imaging system.

FIG. 10 shows an experimental setup with a simple imaging system.

FIG. 11 shows an SPR-dip in angular interrogation.

FIG. 12 shows an SPR-dip in wavelength interrogation.

FIG. 13 shows the relationship between the incident angle for at the SPRcondition and wavelength for gold (i.e. the dispersion relation). Twoprism materials are shown, BK7 and SF11.

FIG. 14 shows the reflectance versus effective refractive index, at thesensor surface, for gold at three different wavelengths at an incidentangle of 67 degrees.

FIG. 15 shows the sensitivity versus effective refractive index, at thesensor surface, for gold at three different wavelengths at an incidentangle of 67 degrees. The curves in FIG. 15 are the derivatives of thecurves in FIG. 14.

FIGS. 16 a-c shows reflectance images for an experimental setup at anincident angle of 68° and wavelengths 634, 692 and 751 nm, with gold assensor metal layer and water as dielectricum.

The invention is now illustrated by description of embodiments withreference to the drawings and experiments, but it should be understoodthat the invention is not limited to the specifically disclosedembodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a two-dimensional imaging surface plasmonresonance apparatus which comprises a sensor surface layer of aconductive material that can support a surface plasmon, a source ofelectromagnetic beams of two or more wavelengths that illuminate atwo-dimensional surface area from either the front or the backside ofthe sensor surface layer, and a detector for simultaneous, or pseudosimultaneous, detection of two or more wavelengths of reflectedintensities from the two-dimensional surface area, providing two or moretwo-dimensional images of the surface area, the two-dimensional imagesbeing a function of the effective refractive index at each point on thesurface area.

In an embodiment of the apparatus of the invention the conductivematerial is a free electron metal, such as gold, silver or aluminum. Thesensor surface layer may be a grating.

In a preferred embodiment of the invention a prism is provided as asupport for the sensor surface layer. The sensor surface layer may besupported on a planar transparent substrate plate, such as glass andplastics, optically attached to the prism, preferably by an indexmatching fluid, gel or glue.

The light source used in the apparatus of the invention may be selectedfrom the group consisting of a) one or more monochromatic light sources,such as light emitting diodes or lasers, b) a glowing filament lamp,such as a Tungsten lamp, and c) a charge discharge lamp, such as a Xenonor Mercury lamp.

In an embodiment of the invention, the light from the light source iscoupled into the sensor surface layer by a lens, fiber optics, or amirror.

In another embodiment the light source provides a variable incidentangle.

In yet another embodiment the light from the light source is collimated.

In still another embodiment the light of different wavelengths from thelight source are impinging on the sensor layer, and by a rotatingfilter, pseudo-simultaneous impinging on the detector, which issynchronized to said rotating filter. The rotating filter can be placedanywhere in the optical path between the light source and the photodetector, i.e. before and after the sensor surface.

The detector used in the apparatus of the invention may be selected fromthe group consisting of a two dimensional array camera, charge coupleddevice (CCD), charge injection device (CID), photo diode array detector(PDA), photomultiplier and a CMOS sensor.

In an embodiment of the invention the detector has a mosaic filter.

In another embodiment two or more detectors are provided, and these arefitted with beam splitters and filters, such as interference filters, toenable measurement of different spectral properties.

In yet another embodiment the filter(s) is(are) adjustable.

In still another embodiment the detector(s) is(are) connected via anoptical fiber bundle.

In a preferred embodiment the detector is a photographic film.

The apparatus of the invention may have a lens system, such as fixedfocal length or a zoom, to magnify or reduce the image.

In a most preferred embodiment the apparatus operates with wavelengthsat or close to the highest slope of the dip, either reflectance versuswavelength or reflectance versus the effective refractive index seen bythe surface plasmon.

In a further embodiment of the invention, the light that hits thedetector is p-polarized by a polarizer.

In another preferred embodiment the two-dimensional images put togetherresult in a color image.

The invention will now be described with reference to the drawings.

FIG. 1 a illustrates one embodiment of the apparatus of the inventionwherein a collimated input beam 110 emanates from an illumination system100 onto a sensor unit 200, preferably a prism (equilateral, rightangle, hemispherical or aspherical) 210. The reflected light from saidsensor unit is projected on an imaging system 500.

FIG. 1 b illustrates schematically an apparatus of the invention with acomputer 900 and an analog to digital converter 800 connected to theimaging system 500.

FIG. 2 a shows the prism 210 of FIG. 1 a onto which a metal film 220 hasbeen evaporated (thermally or by sputtering). The prism 210 istransparent (glass or a polymer). A glass prism can be made of highrefractive index e.g. flint glass or standard glass e.g. crown glass. Apolymer prism can be made of non-crystalline plastics likepolymethylmethacrylate, polycarbonate, styrene, SAN, glycol modifiedPET, etc. The metal film sensor is preferable gold if high chemicalresistance is wanted. The metal film sensor can be silver if highsensitivity is preferred, but chemical resistance is not critical. Themetal film is preferably evaporated on an evaporated (thermally orsputtered) adhesion layer 230. The adhesion layer 230 is preferablychromium or titanium. The adhesion layer is typically 0.5 nm thick. Theadhesion layer is normally not a totally covering film due to thelimited thickness. A too thick adhesion layer will lower the sensitivityof the apparatus. The thickness of said gold film is typically 45 to 50nm. The thickness of said silver film is typically 56 nm. The metallayer 220 may have the form of a pattern. The pattern can be made byreactive etching using a photo lithography process. The metal layer maybe covered by a linker layer 240. The linker layer can be an alkanethiol. The alkane thiol may function as a handle to specific molecules250 a, 250 b, etc. The attached specific molecules can have an affinityto other species 260 a, 260 b, etc. The linker layer 240 can be in theform of a pattern. The metal film can be provided with a pattern by useof an inert film 270. The inert film can be photosensitivebenzocyclobutenes (photo-BCB).

FIG. 2 b shows the base of the prism 210, or transparent substrate 300(FIG. 2 d).

FIG. 2 c shows the prism 210 onto which a flow cell 280 has be attached.To avoid leakage, a seal 290 can be inserted between the prism 210 andthe flow cell 280. The flow cell can be made of a plastic material(polymethylmethacrylate, polycarbonate, styrene, polyvinylchloride,polyetheretherketone, polyamide etc.). The flow cell can be fitted to aflow system 295. The flow system may comprise a pump 297 (e.g. syringepump or peristaltic pump), valves and tubing, as illustrated in FIG. 1b.

FIG. 2 d shows the prism 210 with a metal film 220 evaporated on atransparent substrate 300. The substrate may be a plastic material,glass or semiconductor. The substrate 300 has preferably the same orsimilar optical constants as the prism 210. To obtain good opticalcontact between said prism and said transparent substrate, an opticalinterface 310 is used. The optical interface 310 should have opticalconstants equal to or close to those of said prism and said transparentsubstrate. The optical interface may be an index matching fluid, a gel,or a glue.

FIG. 3 shows an optical system denoted Otto configuration 320. Said Ottoconfiguration utilizes an air or liquid gap 330 to evanescently excitethe surface plasmon.

FIG. 4 illustrates a setup wherein the multi wavelength SPR can beexcited by a corrugated metal film (grating) 340. In this case the metalfilm sensor does not need to be thin.

Referring to FIG. 1 a, the illumination system 100 comprises a lightsource 120. Said light source can be a glowing filament, e.g. atungsten-halogen lamp, or an arc discharge lamp, e.g. Xenon lamp, orlasers, e.g. diode lasers, dye lasers etc, or light emitting diodes(LEDs). Radiation from said light source is collected by a lens system130, which creates a collimated (parallel) light beam 110. The lenssystem 130 can be a positive lens, e.g. f=150 mm, or a condenser system,or a more sophisticated lens system. At some point in the beam of saidillumination system a polarizer can be inserted 140. Said polarizer canbe a dichroic sheet, Glan-Thompson polarizing prisms, Glan-Taylorpolarizing prism or Wollaston prisms. The polarizer or polarizingequipment shall transmit light parallel to the plane of incidence(p-polarized or transverse magnetic, TM). The use of a polarizer willimprove performance, but is not necessary. A surface plasmon can only beexcited by p-polarized light, hence light polarized transverse the planeof incidence (s-polarized or transverse electric, TE) will be reflectedat the sensor surface. Absence of a polarizer will hence decrease thedepth of the SPR dip, which may be a disadvantage, due tonon-informative signal added to the informative SPR signal at theimaging system 500.

The imaging system 500 utilizes at least one area detector 510, whichcan be a photographic film, e.g. a color film (negative or diapositive), an electronic photo device, e.g. photo diode array, chargecoupled device (CCD), charge injection device (CID), CMOS array etc. Thearea detector 510 may be a color device. The color device may include amosaic color filter.

FIG. 5 illustrates an imaging system 500 that consists of more than onearea detector 510 a, 510 b, 510 c, one for each wavelength. In thiscase, a reflected light beam 490 is divided by beam splitters 550 andcolor filters 560 a, 560 b, 560 c, in the same way as used in commercial3 CCD video cameras. The color filters can either be narrow andnon-overlapping in wavelength, or be broad with overlapping wavelengths.

FIG. 6 illustrates a setup for improving image quality by use of animaging lens 520. The imaging lens 520 can be an ordinary focusingcamera lens, and it can be further improved by adding a cylindrical lens530. Image restitution is possible by adding a secondary prism 540,cylinder lens or by tilting the area detector.

FIG. 7 shows an embodiment wherein the incident angle of the light beam110 can be altered by rotating the illumination system 100 with respectto the prism 210. The imaging system 500 is rotated by the same amountas the prism 210, but in the other direction. The rotation can beperformed by a goniometer, i.e. a θ, 2θ system, where the illuminatingsystem is fixed and the prism is rotated θ and the imaging system isrotated 2θ.

FIG. 8 a shows an embodiment wherein the multi-wavelength featuredescribed in relation to FIG. 7 can be fulfilled in a pseudosimultaneously manner with a filter device 600. The filter device 600can be a rotating filter wheel as illustrated in FIG. 8 b. The rotatingfilter 600 can be a band pass filter 610 a, 610 b, 610 c, etc. Said bandpass filter can be an interference filter. Another method to changewavelength is to use an electro-optical device, e.g. a Fabry-Perot cell.Change of filter is synchronized with the detector.

The two-dimensional imaging surface plasmon resonance apparatus of theinvention can be oriented in any direction (vertical, horizontal or anyangle between). The sensor surface can be faced upwards, downwards orany arbitrary direction in space.

DESCRIPTION OF EXPERIMENTS

It is shown how SPR imaging can be performed with a color camera. Thecamera allows simultaneous intensity measurements at differentwavelengths, which will increase the dynamic range and increase thesensitivity and accuracy over a larger range of the refractive index ofthe sensing medium. The sensing medium can be a 3-dimensional bulkmaterial or a 2-dimensional adlayer. For the latter, the thicknesschange can be monitored if the refractive indices of both the adlayerand the surrounding medium are known.

For an SPR apparatus working in angular interrogation (FIG. 11,reflectance versus incident angle) simulations (with Fresnel equations)and measurements show that the resonance angle θ_(sp) increases Δθ_(sp)for increasing effective refractive index, e.g. by an adlayer formation(case 2 in FIG. 11). Case 1 in FIG. 11 is without an adlayer, i.e. alower effective refractive index is seen by the sensor. Simulations(Fresnel equations) and measurements show that the resonance wavelengthincreases with increasing effective refractive index (e.g. adlayerformation) for an apparatus working in the wavelength interrogation(FIG. 12). The conditions used in the example is a gold film ofthickness, d_(m), equal to 50 nm, an incident angle, θ, equal to 67°,and a prism 210 made of BK7 glass. The curves in FIG. 12 illustrate thereflectance versus wavelength for effective refractive indices, n_(a),from 1.33 to 1.37.

Referring to FIG. 13 and Fresnel calculations, the SPR dip-valley(minimum reflection) will move in a right upward direction uponincreasing effective refractive index, n_(a), of the dielectricum, e.g.an adlayer formation. The curvature of the dip-valley is an effect ofthe SPR-dispersion relation emanating from the dispersion of the metal(i.e. change of dielectric constant as a function of wavelength). Theincident angle, wavelength, refractive index of the prism 210 andeffective refractive index of the sample for the SPR imaging equipmentare coupled (due to the dispersion relation of the surface plasmon).Curves from two different setups are shown in FIG. 13, one correspondsto a prism 210 made of BK7 glass (refractive index approximately equalto 1.5), and the other corresponds to a prism 210 made of SF11 glass(refractive index approximately equal to 1.5). The incident angle is beplotted versus the refractive index of the sample (FIG. 14) for the sameconditions as in FIG. 12, using three wavelengths (700, 800, and 900nm). The incident angle and wavelength is preferably chosen so that thederivative (FIG. 15) of the reflectance versus effective refractiveindex (FIG. 14) is maximized. The derivatives of the reflectance versuseffective refractive index for an example with a 50 nm gold film, at anincident angle of 67°, for three different wavelengths (700, 800, and900 nm), and different effective refractive indices are shown in FIG.15.

An example of a sensor surface that has individual sensor spots on agold film 220 is shown in FIG. 16 for wavelengths 634, 692 and 751 nm,at an incident angle of 68° and a prism made of BK7 glass. The effectiverefractive index of the sample is approximately 1.33.

EXAMPLE 1

FIG. 9, which illustrates one embodiment of the two-dimensional imagingsurface plasmon resonance apparatus of the invention, utilizes a whitelight source comprising a 300 W Xenon Arc lamp 800 (Oriel Inc.,Stratford, Conn., USA), in a housing 810 (Oriel) containing a F/1condensing lens assembly 820 (Oriel). Collimated light is focused by apositive lens, f=150 mm 830 (Oriel), onto a 400 m pinhole 840 (MellesGriot Inc.). A second lens, f=150 mm 850 (Melles Griot), creates acollimated light beam 875, which is filtered by a filter wheel 860consisting of three interference filters of center wavelength 634, 692and 751 nm respectively. All filters have a bandwidth of 10 nm. Saidcollimated beam is plane polarized by a dichroic sheet polarizer 870(Melles Griot). The collimated light 875 impinges on an equilateralprism 880 made of BK7 glass (Melles Griot). Onto said prism is a glasssubstrate 883 attached by an index matching fluid (Cargille Inc.). Theglass substrate contains the metal film supporting the surface plasmonresonance. The reflected light 877 from said prism 880 is directed to aright angle prism 890 made of BK7 glass (Melles Griot). Said prism 890restitutes the image. A cylinder lens, f=100 mm, 900 is put between thesaid right angle prism 890 and a camera lens, Nikon micro f=60 mm,f/2.8, 910. The image from the sensor surface is projected on a CCDcamera 920 (Orbis 2, Spectra Source Inc.)

EXAMPLE 2

FIG. 10, which illustrates another embodiment of the two-dimensionalimaging surface plasmon resonance apparatus of the invention, utilizes awhite light source comprising a 300 W Xenon Arc lamp 800 (Oriel Inc.,Stratford, Conn., USA), in a housing 810 (Oriel) containing a F/1condensing lens assembly 820 (Oriel). Collimated light is focused by apositive lens, f=150 mm 830 (Oriel), onto a 400 m p inhole 840 (MellesGriot Inc.), A second lens, f=150 mm 850 (Melles Griot), creates acollimated light beam 875, which is filtered by a filter wheel 860consisting of three interference filters of center wavelength 634, 692and 751 nm, respectively. All filters have a bandwidth of 10 nm. Saidcollimated beam is plane polarized by a dichroic sheet polarizer 870(Melles Griot). The collimated light 875 impinges on an equilateralprism 880 made of BK7 glass (Melles Griot). Onto said prism is a glasssubstrate 883 attached by an index matching fluid (Cargille Inc.). Theglass substrate contains the metal film supporting the surface plasmonresonance. The reflected light 877 from said prism 880 is projected on aCCD camera 920 (Orbis 2, Spectra Source Inc.) The camera is tilted torestitute the image.

All the references cited in this specification are included herein byreference.

REFERENCES

-   [1] E. Kretschmann, Die Bestimmung Optischer Konstanten von Metallen    Durch Anregung von Oberflächenplasmaschwingungen, Z. Physik, Vol.    241, (1971), 313-324.-   [2] E. Yeatman and E. Ash, Surface Plasmon Microscopy, Electronics    Letters, Vol. 23, (1987), 1091-1092.-   [3] B. Ivarsson, Analytical Method and Apparatus, Patent EP958494A1.-   [4] B. Ivarsson, Analytical method and apparatus, Patent    WO9834098A1.-   [5] B. P. Nelson, A. G. Frutos, J. M. Brockman and R. M. Corn,    Near-Infrared Surface Plasmon Resonance Measurements of Ultrathin    Films. 1. Angle Shift and SPR Imaging Experiments, Analytical    Chemistry, Vol. 71, (1999), 3928-3934.-   [6] T. Turbadar, Complete Adsorption of Light by Thin Metal Films,    Proc. Phys. Soc. Lond., Vol. 73, (1959), 40-44.-   [7] A. Otto, Excitation of Nonradiative Surface Plasma Waves In    Silver by the Method of Frustrated Total Reflection, Z. Physik, Vol.    216, (1968), 398-410.-   [8] B. Liedberg, C. Nylander and I. Lundström, Surface Plasmon    Resonance For Gas Detection and Biosensing, Sensors and Actuators,    Vol. 4, (1983), 299-304.-   [9] E. M. Yeatman and E. A. Ash, Surface plasmon scanning    microscopy, Proceedings of SPIE, Vol. 897, (1988), 100-107.-   [10] C. E. Jordan and R. M. Corn, Surface Plasmon Resonance Imaging    Measurements of Electrostatic Biopolymer Adsorption onto Chemically    Modified Gold Surfaces, Analytical Chemistry, Vol. 69, (1997),    1449-1456.-   [11] C. E. Jordan, A. G. Frutos, A. J. Thiel and R. M. Corn, Surface    Plasmon Resonance Imaging Measurements of DNA Hybridization    Adsorption and Streptavidin/DNA Multilayer Formation at Chemically    Modified Gold Surfaces, Analytical Chemistry, Vol. 69, (1997),    4939-4947.-   [12] B. Rothenhäusler and W. Knoll, Surface-plasmon microscopy,    Nature, Vol. 332, (1988), 615-617.-   [13] W. Hickel, B. Rothenhäusler and W. Knoll, Surface plasmon    microscopic characterization of external surfaces, Journal of    Applied Physics, Vol. 66, (1989), 4832-4836.-   [14] B. Rothenhäusler and W. Knoll, Interferometric determination of    the complex wave vector of plasmon surface polaritons, Journal of    Optical Society of America B, Vol. 5, (1988), 1401-1405.-   [15] U. Fernandez, T. M. Fischer and W. Knoll, Surface-plasmon    microscopy with grating couplers, Optics Communications, Vol. 102,    (1993), 49-52.

1. A two-dimensional imaging surface plasmon resonance apparatus whichcomprises a sensor surface layer of a conductive material that cansupport a surface plasmon, a source of electromagnetic beams of two ormore wave lengths that are collimated and illuminate a two-dimensionalsurface area from either the front or the backside of the sensor surfacelayer, and a detector for simultaneous, or pseudo simultaneous,detection of two or more waveiengths off reflected intensities from thetwo-dimensional surface areas, providing two or more two-dimensionalimages of the surface area, the two-dimensional images being a functionof the effective refractive index at each point on the surface area. 2.The apparatus according to claim 1, wherein the conductive material is afree electron metal.
 3. The apparatus according to claim 2, wherein thefree electron metal is selected from the group consisting of gold,silver and aluminum.
 4. The apparatus according to claim 1, wherein thesensor surface layer is a grating.
 5. The apparatus according to claim1, wherein a prism is provided as a support for the sensor surfacelayer.
 6. The apparatus according to claim 5, wherein the sensor surfacelayer is supported on a planar transparent substrate plate, opticallyattached to the prism.
 7. The apparatus according to claim 6, the planartransparent substrate plate is selected from glass and plastics, and theoptical attachment is by an index matching fluid, gel or glue.
 8. Theapparatus according to claim 1, wherein the light source is selectedfrom the group consisting of a) one or more monochromatic light sources,b) a glowing filament lamps, and c) a charge discharge lamp.
 9. Theapparatus according to claim 8, wherein the light source a) is selectedfrom the group consisting of light emitting diodes and lasers, the lightsource b) is a Tungsten lamp, and the light source c) is a Xenon orMercury lamp.
 10. The apparatus according to claim 1, wherein the lightfrom the light source is coupled into the sensor surface layer by alens, a fiber optics, or a mirror.
 11. The apparatus according to claim1, wherein the light source provides a variable incident angle.
 12. Theapparatus according to claim 1, wherein the light of differentwavelengths from the light source are pseudo-simultaneous impinging onthe sensor layer by a rotating filter and are synchronized to thedetector.
 13. The apparatus according to claim 1, wherein the detectoris selected from the group consisting of a two dimensional array camera,charge coupled device (CCD), charge injection device (CID), photo diodearray detector (PDA), photomultiplier and a CMOS sensor.
 14. Theapparatus according to claim 1, wherein the detector has a mosaicfilter.
 15. The apparatus according to claim 1, wherein two or moredetectors are provided, and these are fitted with beam splitters andfilters, such as interference filters, to enable measurement ofdifferent spectral properties.
 16. The apparatus according to claim 14,wherein the filter(s) is(are) adjustable.
 17. The apparatus according toclaim 1, wherein the detector(s) is(are) connected via an optical fiberbundle.
 18. The apparatus according to claim 1, wherein the detector isa photographic film.
 19. The apparatus according to claim 1, wherein alens system, such as fixed focal length or a zoom, is provided tomagnify or reduce the image.
 20. The apparatus according to claim 1,wherein the apparatus operates with wavelengths at or close to thehighest slope of the dip, either reflectance versus wavelength orreflectance versus the effective refractive index seen by the surfaceplasmon.
 21. The apparatus according to claim 1, wherein the light thathits the detector is p-polarized by a polarizer.
 22. The apparatusaccording to claim 1, wherein the two-dimensional images put togetherresult in a color image.